Apidologie 41 (2010) 568578

c INRA/DIB-AGIB/EDP Sciences, 2010


DOI: 10.1051/apido/2010004

Available online at:

www.apidologie.org

Original article

Chemical cues in the host-seeking behaviour of tracheal

mites (Acarapis woodi) in honey bees (Apis mellifera
mellifera)*
John B. Mcmullan1 , Patrizia dettorre2 , Mark J.F. Brown3
1

School of Natural Sciences, Department of Zoology, University of Dublin Trinity College, Dublin 2, Ireland
2
Centre for Social Evolution, Department of Biology, University of Copenhagen, Denmark
3
School of Biological Sciences, Royal Holloway, University of London, Egham, TW20 0EX, UK
Received 18 June 2009 Revised 23 September 2009 Accepted 29 November 2009

Abstract The tracheal mite, Acarapis woodi, is a widespread parasite of honey bees that can lead to colony
death. A key determinant of colony mortality is the prevalence of the parasite within a colony. Prevalence
depends upon transmission rates, which vary depending upon the age of potential hosts. Honey bee cuticular
hydrocarbons change with age, and have been suggested to be the cue used by tracheal mites to determine
whether they move to a potential host. Recently, honey bees pupated at a reduced temperature were found
to have a significantly higher susceptibility to tracheal-mite infestation. In this study experiments were conducted to determine whether this susceptibility was due to a change in chemical cues. Cuticular compounds
were significantly dierent between bees pupated at 34 C and 30 C, as predicted. However, behavioural
tests showed conclusively that the increased susceptibility of bees pupated at the lower temperature could
not be explained by these chemical dierences. Other factors, such as changes in bee physiology, reduced
mobility and cognitive ability, that have been shown to be caused by reduced pupation temperature, are suggested as potential mechanisms. Interestingly, these experiments provide evidence that tracheal mites only
respond to gustatory and not olfactory cues. The implications of this for the behavioural strategies adopted
by mites to maximise safe transmission, and a new model for mite transfer are discussed.
Apis mellifera / Acarapis woodi / tracheal mite / mite migration / pupation temperature

1. INTRODUCTION
The tracheal mite, Acarapis woodi, has
been associated with widespread damage to
honey bee colonies since the early 1900s
(Bailey, 1961; Adam, 1987; Eischen et al.,
1989; Otis and Scott-Dupree, 1992). The mite
is an obligate parasite of the honey bee and
completes its lifecycle entirely within the tracheae of the honey bee except for the transitionary period when it is moving to a new,
honey bee host.
It has been known for some time that
the susceptibility of bees to tracheal mites is
Corresponding author: J.B. McMullan,
jmcmullan@eircom.net
* Manuscript editor: Peter Rosenkranz

at its highest at time of emergence and declines rapidly with age. During normal colony
conditions when brood and young bees are
present, bees more than 4 days old are seldom
invaded by new mites (Morgenthaler, 1931;
Bailey, 1958; Lee, 1963; Giordani, 1977; Gary
and Page, 1987). Susceptibility is also dependent on the strain of bee (Gary and Page,
1987) and this has been shown to be influenced to a large extent by the grooming ability of the bees (Danka and Villa, 1998, 2005;
McMullan and Brown, 2006). It has also been
demonstrated that reduced pupation temperature causes honey bee susceptibility to increase (McMullan and Brown, 2005). This increase in susceptibility may play a key role
in the process whereby tracheal mites cause
honey bee colony mortality (McMullan and

Article published by EDP Sciences

Host-seeking behaviour of Acarapis woodi

Brown, 2009). Changes in autogrooming cannot explain this pupation temperature eect
(McMullan and Brown, 2006) and hence the
question remains as to what is the principal
mechanism behind this increased susceptibility. What causes honey bees pupated at a lower
than normal temperature to have a higher number of foundress female mites migrate to their
tracheae? One possible explanation for this
dierential susceptibility may be the influence
of chemical cues.
Hydrocarbons make up the major part of
the lipid layer on the outer cuticular surface
of honey bees. Branched and saturated hydrocarbons decrease rapidly from the time of
emergence of new callow bees (Francis et al.,
1989) and this may be used by mites to identify young bees. Phelan et al. (1991) concluded
that the specific attractiveness of young callow
bees to tracheal mites was determined by their
cuticular compounds, and demonstrated a significant eect in three out of four honey bee
test colonies. Using a similar methodology,
vanEngelsdorp and Otis (2001) undertook an
experiment to identify if cuticular compounds
had a role in the dierential resistance of different bee strains to infestation by the tracheal
mite. While the results demonstrated that the
mites had a preference for some bee colonies
as opposed to others there was no consistency
based on colony susceptibility/resistance.
Phelan et al. (1991) used hexane to extract the cuticular compounds from test bees
and employed a two-choice bioassay to identify mite preference. The hexane extracts were
applied to two filter papers (1 cm diameter),
4 mm apart on a plastic slide and the female
mite was placed in the centre. The location of
the mite after a period of 5 minutes was used
to indicate a preference for one extract over
the other. If mites are attracted to the cuticular extract of bees it must also be possible to
elicit a response from a direct whole bee approach. By presenting mites with treated bees
it would be expected that a mite should express in a two-choice bioassay any preference
that was present. Sammataro and Needham
(1996) showed that mites placed on the thoraces of treated bees demonstrated treatmentdependent responses.

569

Here we ask whether chemical cues determine the increased susceptibility to tracheal
mite infestation of honey bees pupated at a
lower than normal temperature. To do this we
used a two-pronged approach. First, we conducted behavioural choice tests on the mites.
Second, we obtained profiles of the honey bee
surface hydrocarbons using gas chromatography mass spectrometry. We combined the results from these experiments to enhance our
understanding of the dynamics of tracheal mite
transmission within honey bee colonies.
2. MATERIALS AND METHODS
A two-choice experiment was originally set up
along the same lines as that in Phelan et al. (1991),
and vanEngelsdorp and Otis (2001) with the exception that a whole bee was used in place of cuticular
extract.

2.1. Preparation of the test bees

The source honey bees for the experiment were
the control (34 C) and treated bees (30 C) from
the three colonies that were used in the pupationtemperature experiment (McMullan and Brown,
2006). The colonies were of the subspecies Apis
mellifera mellifera and from beekeeper apiaries,
four to eight kilometres apart, in North County
Dublin, Ireland. These callow bees (less than
8 hours old), which were part of the same sample of
bees used in the 2006 experiment, had been frozen
and held at 30 C. Bees pupated at 34 C and 30 C
were removed from the freezer and held at room
temperature for 20 minutes to allow them to dry o.
The bees to be hexane washed were put in a glass
container for 10 minutes with 4 mL of hexane. The
container was agitated at the beginning and end of
the period after which the bees were removed and
allowed to dry-o at room temperature.

2.2. Initial two-choice arrangement

A plastic slide cover had the centre line marked
and a 4 mm diameter hole (similar in diameter to
a bee thorax) was punched on each side of the
centre line, the holes being 4 mm apart (Fig. 1a).
The control and treated bees were inserted from below into the holes and wedged to the level of the

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J.B. McMullan et al.

(a)

2.3. Selecting female test mites

4 mm
4 mm dia. holes

(b)
Thoraces with heads
removed
Expanded
view
C

Plastic slide
cover
C

4 mm dia.
2mm

Pleural hair
overlapping

C - control bee; T - treated bee

Figure 1. (a) Line drawing of the initial two-choice

test arrangement similar to that used by Phelan et al.
(1991) with the exception that a whole bee is used
in place of extract of cuticular compounds. The
slide cover with 4 mm holes rests on a wire support
with the bees inserted from below. (b) Line drawing of modified two-choice arrangement with bees
mounted using coaxial straws, the inner straw passing through the 4 mm dia. holes in a plastic slide
cover. The two bees were inserted into the inner
straws, wider straws on the outside went over the
wings and legs and the thoraces were pushed together to allow the plural hair to overlap. The slide
cover rests on a wire support.

The mites were harvested from a host colony

with a mite prevalence of >70%. Flying bees in
groups of about 40 were collected from the landing board of the hive using a vacuum collector.
They were held, awaiting dissection, in wire cages
in an incubator and fed water and 50% (w/w) sugar
syrup. The live female mites for the two-choice testing were obtained by removing the bees head and
pronotum thereby exposing the tracheae. To avoid
injuring the mites, a piece of cuticle was cut around
the spiracle end of the trachea and this was removed
intact with the infested trachea under a stereomicroscope at 60100X magnification and placed on a
glass slide. Some salivary gland tissue was also extracted to enable the trachea to adhere to the glass
slide. No more than five infested tracheae were removed simultaneously to minimise the time that the
mites were outside of the dissected bee. An eyelash
attached to a thin wooden probe was used to lift the
mites. The female mites were lifted from a questing position on the hair attached to the cuticle at
the spiracle end of the trachea or by gently opening
the trachea with a dissecting needle and removing
a mite. Only young (lightly bronzed) female mites
and lightly pregnant mites were used. Old heavily
pregnant mites tended to lack mobility and stayed
fixed in one position in the test area.

2.4. Initial two-choice test

wing roots. The plastic slide cover with bees attached was then placed horizontally on a wire support. The bees were in a vertical axis, aligned laterally to each other and facing in opposite directions.
The tests were undertaken blind. Giordani (1967)
demonstrated that the highest survival rate for tracheal mites on bee pupae was at a temperature of
28 C and relative humidity (RH) of 70%. The experiment was conducted in an ambient temperature
of 28 ( 1) C and (RH) of 68 ( 5)%. These conditions were maintained in a confined laboratory
using a humidifier and meant that the removal of
mites from the infested bees, their transfer and the
experiment itself were undertaken in stable conditions causing the minimum environmental shock to
the mites.

The experiments of Phelan et al. (1991) had significant responses in all cases where cuticular hydrocarbon extract from a bee of any age was tested
against a hexane control. In order to test for a similar response, the 34 C pupated bees as a control
were tested against 34 C pupated bees that had
been washed in hexane for 10 minutes. The test
was repeated for the 30 C (unwashed vs. washed)
as well as 34 C vs. 30 C (both unwashed). The
test was carried out on one colony, colony 2, as this
colony showed the greatest change in susceptibility
when pupated at 30 C in the pupation-temperature
experiment (Fig. 2). The selected female mite was
placed in the centre position between the test bees
and its location was scored after 5 minutes as being
on the control or treated bee or on the slide cover
as a no choice. A total of 16 pairs were tested for
each case. Since this approach did not elicit any observable response the alternative modified approach
below was employed.

Host-seeking behaviour of Acarapis woodi

1.2

34C (Control)
30C

Female Mites per bee

0.8

0.6

0.4

0.2

0
1

Colonies

Figure 2. Mite abundance levels (mean female

mites per bee) for the bees pupated at 34 C and
30 C, from the three colonies, after a period
of seven days in the inoculation cages (modified
from McMullan and Brown, 2006). Statistically significant dierence within colony is indicated by
asterisk.

2.5. Modified two-choice test

An alternative two-choice approach was designed to enable the mites to be in contact simultaneously with cuticular compounds from the control and treated bees, and to make their choice using
the gustatory sensors on their legs (Waladde and
Rice, 1982). The head was removed from each bee
to improve the visibility of the thorax, when viewed
through the microscope from the top, and excess
body fluid was removed with blotting paper to prevent it staining the hairs on the thorax and aecting their condition. The bees were mounted in the
vertical axis within short lengths of 4 mm diameter straws spaced 2 mm apart (Fig. 1b). The thinner
straws were inserted into two 4 mm holes that had
been punched 2 mm apart in a plastic slide, and this
held the straws in place.
The bees were pushed abdomen first into the
thinner straws as far as the root of the wings, with
wings and legs on the outside. They were then held
in position with short lengths of wider straws that
went over the inside straws and trapped the wings
and legs in position. The slide cover was placed horizontally on a wire support. The bees were in a vertical axis, aligned laterally to each other and facing in
opposite directions, and were tilted slightly towards
each other to enable the pleural hair on the thoraces
to interlock. The pleural hair was combed with
an eyelash probe to enhance the interlocking of the

571

hair. Separate probes were dedicated to either the

control or treated bees to avoid cross-contamination
of the cuticular compounds. The hair was allowed to
overlap by about a third of its length.
A selected female mite was deposited at the centre of the overlapping pleural hair. Since the female
mites are large relative to the thickness of the pleural hair they were simultaneously in contact with the
hair of both test bees. The mite choice was scored
after 5 minutes by noting the location of the mite.
A no score was registered if the mite had not located itself on one or other of the two bees. The behavioural mode of the mites (questing/seeking, see
below) was noted after 5 minutes and also after a period of about 3 hours. It had been hoped to dissect
bees after the longer period to establish how many
of the mites had migrated into the tracheae. However, after this period dissection was not possible as
dark body fluid had gathered in the tracheae.
It was of some concern that the dead bees would
lose their freshness. To test the experimental protocol 16 pairs of samples of bees from each pupation temperature in colony 2, were presented as
an unwashed vs. washed bee choice. Eight pairs
were tested within three hours of the bees being removed from freezer storage and the second batch
was tested 4 to 6 hours post-removal. The mite
preference for unwashed vs. washed in the first
3 hours was 13:2 respectively and the corresponding choices in the 4 to 6 hour period 8:7. This
would indicate that the decomposing of the bees
cloaked any dierence in bee cuticular compounds
in the two-choice test, as a significant dierence in
choices was demonstrated between the two periods
(2 = 3.96, P < 0.05). As a result it was decided
to test the bees in batches of 4 pairs and the tests
were undertaken within 2 hours of the bees being
removed from storage.
The permutations used to obtain the initial twochoice test for response above were repeated but
this time for all three colonies as opposed to only
colony 2. For each colony 32 two-pair tests were undertaken for the 34 C (unwashed vs. washed) and
30 C (unwashed vs. washed). In the case of 34 C
vs. 30 C (both unwashed), 48 pairs were tested for
each colony.
In the migration process from old to new host
bees two of the mites critical behavioural modes,
questing and seeking, were noted. Hirschfelder
and Sachs (1952) identified questing as the behaviour where the mite crawls up a bee hair (seta),
holds on with a leg and tries to make contact with
any adjacent objects (Fig. 3). Seeking on the other

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Table I. Initial two-choice arrangement. Female tracheal mite choices when presented with 16 pairs
of bees, made up of unwashed (uw) and hexane
washed (w) bees, pupated at 34 C and 30 C
from colony 2. The thoraces of bee-pairs protruded
through holes spaced at 4 mm in a cover slip and the
mites were placed in the centre.
Choice

Figure 3. Photograph of mite in questing mode,

with its right rear leg attached to a bee hair (seta).

hand describes niche-location behaviour on the bee

including finding the entrance to the spiracle.

2.6. Chemical analyses

Samples of frozen callow bees from each of
the three colonies and 34 C & 30 C temperature treatments per colony (six sets of samples in
total) as used in the two-choice experiments, were
analysed to obtain the profiles of their cuticular hydrocarbons. The chemical analysis was undertaken
in the Department of Population Biology, Institute
of Biology, University of Copenhagen, Denmark.
Ten bees were chosen from each of the six samples. Each bee was washed in 500 L of pentane
for 10 minutes. The bee was then removed from
the vial and the solvent allowed to evaporate o
at room temperature for approximately 30 minutes.
The extract was re-diluted in 110 L of pentane
before being analysed with an Agilent Technologies 6890N gas chromatograph (capillary column:
HP5MS 30 m X 250 m X 0.25 m; injector: splitsplitless; carrying gas: helium at 1 mL/min), coupled with a 5975 mass selective detector (70 eV
electron impact ionisation). The temperature programme ranged from 70 C to 280 C at 10 C/min.
and from 280 C to 310 C at 3 C/min., and held at
310 C for 10 min. Injections were performed by an
automatic liquid sampler 7683B. Compounds were
identified on the basis of their mass spectra. The
presence of 29 regularly occurring hydrocarbons
was identified and the area for each peak was calculated for each bee with the software Chemstation
(Agilent Technologies).

Outcome
uw:w
(no choice)
34 C (uw):34 C (w)
1:2 (13)
2:0 (14)
30 C (uw):30 C (w)
34 C (uw):30 C (uw) 1:2 (13)
All choices
4:4 (40)

2 test (P value)
choice:no choice
6.25 (P = 0.01)
9.0 (P < 0.01)
6.25 (P = 0.01)
21.3 (P < 0.01)

2.7. Statistical analyses

The data from the two-choice tests established,
(i) if the female mites made a choice, (ii) the preferences between unwashed and washed bees and
(iii) the preferences between 34 C and 30 C treatments. All of the data were analysed using chisquare tests. The chemical profiles of the bees from
the three colonies and two temperature treatments
were compared using a multivariate analysis. Peak
areas were transformed using a Z-transformation
(Aitchison, 1986) and then analysed with a principal component analysis (PCA) to reduce the number of variables, which were subsequently analysed
using discriminant analysis (DA) in STATOSTICA
(StatSoft).

3. RESULTS
3.1. Two-choice tests
The initial two-choice arrangement with the
test bees 4 mm apart did not elicit a meaningful response (Tab. I). After release, the mites
generally moved in circles relatively close to
the centre of the slide or moved, at what appeared to be at random, in a particular direction. On occasions when a mite moved close to
one of the bees there was no evidence that the
mite had any awareness of the proximity of the
bee (for example, mites did not turn towards
the bee). Consequently, the number of individual choices made by the mites (that is, the
number of times that they walked onto a bee)

Host-seeking behaviour of Acarapis woodi

573

Table II. Modified two-choice arrangement. Female tracheal mite choices when presented with 32 pairs
of unwashed (uw) and hexane washed bees (w), pupated at 34 C and 30 C from the three colonies. The
plumose hair of the bee-pairs was overlapping and the mites were placed in the centre.
Colony

Choice
uw:w (no choice)

2 test (P value)
choice:no choice

uw:w

Colony 1
34 C

12:17 (3)

21.2 (P < 0.01)

0.86 (P = 0.36)

30 C

16:13 (3)

21.2 (P < 0.01)

0.31 (P = 0.58)

Total (Col. 1)

28:30 (6)

42.3 (P < 0.01)

0.07 (P = 0.80)

34 C

21:8 (3)

21.3 (P < 0.01)

5.80 (P = 0.02)

30 C

21:9 (2)

24.5 (P < 0.01)

4.80 (P = 0.03)

Total (Col. 2)

42:17 (5)

45.6 (P < 0.01)

10.59 (P < 0.01)

Colony 2

Colony 3
34 C

17:10 (5)

15.1 (P < 0.01)

1.81 (P = 0.17)

30 C

19:9 (4)

22.2 (P < 0.01)

3.57 (P = 0.05)

Total (Col. 3)

36:19 (9)

33.1 (P < 0.01)

5.25 (P = 0.02)

34 C

50:35 (11)

57.0 (P < 0.01)

2.65 (P = 0.10)

30 C

56:31 (9)

63.3 (P < 0.01)

7.18 (P < 0.01)

34 C

38:18 (8)

36.0 (P < 0.01)

7.14 (P < 0.01)

30 C

40:18 (6)

42.2 (P < 0.01)

8.34 (P < 0.01)

All colonies

Col. 2 & 3

was so low as to yield no worthwhile comparisons. In all only 8 choices were made out
of 48 two-pair tests representing a significant
no-choice preference (2 = 21.3, P < 0.01).
The experiment demonstrated that the mites
were quite mobile in this new environment and
those mites that did not find one of the two
bees could still be seen moving on the slide
cover for up to three hours. These results suggest that mites cannot detect dead host bees
from a distance, through olfaction, and thus
we used the second experimental protocol to
examine host-choice behaviour.
When the choices between hosts described
above were presented to the mites in the modified two-choice arrangement (with the interlocking pleural hair) the choice (vs. no choice)
option was significant in all cases, with most
mites choosing a potential host bee (Tabs. II
and III). This is in stark contrast to the initial
experimental protocol. However, the choices

made by mites between hosts varied depending upon host colony (Tab. II).
Mites presented with bees from colony 1
expressed no significant preference for an unwashed as opposed to a washed bee (Tab. II).
In contrast, when presented with potential
hosts from colonies 2 and 3 a significant preference (P < 0.05) was expressed for the unwashed bee in 3 out of 4 cases (Tab. II). There
was also a significant preference for the unwashed bees when data for the treatments were
combined for colonies 2 and 3 (2 = 10.59,
P < 0.01 and 2 = 5.25, P = 0.02, respectively) and when colonies were combined
for treatments 34 C and 30 C (2 = 10.59,
P < 0.01 and 2 = 5.25, P = 0.02, respectively).
The mites did not have a significant preference for 30 C as opposed to 34 C pupated
bees (Tab. III). While the mites did choose
bees pupated at 30 C 26 times compared to

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J.B. McMullan et al.

Table III. Modified two-choice arrangement. Female tracheal mite choices when presented with 48 pairs of
bees pupated at 34 C and 30 C, from the three colonies. The plumose hair of the bee-pairs was overlapping
and the mites were placed in the centre.
Colony

Colony 1
Colony 2
Colony 3
All colonies
Col. 2 & 3

Choice
34 C:30 C
(no choice)
17:26 (5)
19:25 (4)
24:21 (3)
60:72 (12)
43:46 (7)

17 times for 34 C pupated bees for colony 1

(2 = 1.88, P < 0.17) the combined data for
colonies 2 and 3 show a 46:43 non significant
preference for 30 C compared to 34 C pupated bees (2 = 0.10, P < 0.75).
In 81% (271/336) of the bee-pairs undertaken in the modified two-choice arrangement,
the mites were in seeking mode when scoring
was undertaken at the end of 5 minutes. After
three hours less than a quarter of the mites remained on the bees, these being fairly evenly
divided between seeking and questing (39:43).
In a few cases the mites could be seen in a
feeding position on the thorax cuticle or in the
thorax dorsal groove.
3.2. Chemical analysis
The cuticular hydrocarbons on the honey
bee samples were those typical of the species,
i.e. a mixture of linear, branched and doublebonded configurations with chain lengths
ranging from C19 to C33. The chemical profile of bees reared at 30 C is clearly separated from that of bees reared at 34 C by a
discriminant analysis including three colonies
and thus six groups (Fig. 4 Wilks Lambda:
0.0049, F[45,204] = 10.377, P < 0.0001).
The dierence due to the rearing temperature had such an important eect on the bee cuticular hydrocarbons that it masked the eect
of the origin of the colony. Indeed, a discriminant analysis based only on two groups (bees
reared at 30 C and 34 C), independent of the
colony, is highly significant (Wilks Lambda:
0.1669, F[9,49] = 27.171, P < 0.0001).

2 test (P value)
choice:no choice
34 C:30 C
30.1 (P < 0.01)
33.3 (P < 0.01)
36.7 (P < 0.01)
100.0 (P < 0.01)
169.0 (P < 0.01)

1.88 (P = 0.17)
0.82 (P = 0.37)
0.20 (P = 0.66)
1.09 (P = 0.30)
0.10 (P = 0.75)

4. DISCUSSION
4.1. Increased susceptibility due
to reduced pupation temperature
We found no evidence that cuticular compounds determine the increased susceptibility
to tracheal mite infestation of honey bees pupated at 30 C compared to 34 C (Tab. III).
Mites that migrated to a bee in the two-choice
experiment did not dierentiate between a bee
pupated at 34 C compared to one pupated at
30 C. While the chemical analysis (Fig. 4) indicated there was a clear distinction between
the cuticular hydrocarbons in the two temperature treatments this did not appear to be used
by the mites in their host choice.
It was demonstrated in earlier research that
reduced autogrooming was only implicated
to a limited extent in the increased susceptibility due to reduced pupation temperature
(McMullan and Brown, 2006). In this paper
there was no evidence that the mites use cuticular hydrocarbons to express a preference for
callow bees pupated at the lower temperature
(Tab. III). Hence since there is no evidence in
this case that honey bee dierential grooming
or cuticular hydrocarbons are important factors in determining honey bee susceptibility,
the relative physiology of the bees may be the
factor that causes more founding female mites
to migrate to bees pupated at a lower temperature. There is some evidence to support this
conclusion. First, the relationship between bee
susceptibility and bee age (the susceptibility
curve) has a high initial susceptibility for new
callow bees and this rapidly diminishes to near

Host-seeking behaviour of Acarapis woodi

575

Figure 4. Plot of the first two roots of the discriminant analysis based on the surface hydrocarbons from the
three colonies of bees with temperature treatments 34 C and 30 C (n = 10 per treatment per colony). The
hydrocarbon profiles of bees from the two temperature treatments show a significant separation both when
the colony of origin is included in the analysis (Wilks Lambda: 0.0049, F[45,204] = 10.377, P < 0.0001) and
when the analysis is based only on two groups, i.e. 34 C and 30 C reared bees (Wilks Lambda: 0.1669,
F[9,49] = 27.171, P < 0001).

zero at 4 to 5 days old. Data from previous experiments (McMullan and Brown, 2005) suggest that the susceptibility curve for bees pupated at lower temperatures, whilst declining
to near zero at 4 to 5 days old, was above
the curve for bees pupated at normal temperatures. Thus, increased susceptibility was due
to higher initial susceptibility, rather than an
extension of the susceptibility period. Second,
Tautz et al. (2003) showed that bees pupated
at a low temperature are less active and have a
significantly reduced dance and cognitive ability. They also suggested, that the temperature at which pupae are raised will influence
their behavioural performance as adults and
may determine the tasks they carry out best inside and outside the hive. Furthermore, Jones
et al. (2005) demonstrated that reduced pupation temperature aected the short term learn-

ing and memory of the bee. This may result in

relatively reduced mobility in these bees. Finally, Pettis et al. (1992) demonstrated that reduced bee mobility coincides with increased
mite migration, which occurs almost exclusively at night-time when the bees are in a
quiescent mode. Together these studies indicate that changes in bee physiology, in the
form of reduced mobility and cognitive ability, are likely to be the mechanism whereby reduced pupation temperature causes increased
bee susceptibility.
4.2. Role of gustatory senses during
migration
Our work also gives insight into the nature of mite migration. In our experiments,

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J.B. McMullan et al.

the mites did not respond to bees that were

dead even when they were in close proximity to them. It was only when they came
in contact with the bees that there was any
response. We do acknowledge that the test
protocol essentially registers the mites initial choice. However, the modified arrangement did allow for the mite to be in contact with the pleural hair of both host bees
and hence there was a comparative element
in the mites choice. Thus there is evidence
that the mites are not stimulated through their
olfactory senses but through their gustatory
senses requiring direct contact between the
mite and the potential host bee (Tabs. I and II).
Hirschfelder and Sachs (1952) have shown
that mites responded to contact and not the
scent of old or young bees while Sammataro
and Needham (1996) have demonstrated that
mites detect the dierent thoracic conditions
only if placed on the host. Thus while the findings in our experiments are consistent with
the work of Hirschfelder and Sachs (1952),
and Sammataro and Needham (1996), it is
not clear whether the findings of Phelan et al.
(1991) and vanEngelsdorp and Otis (2001)
demonstrate a response based on an olfactory
or a gustatory sensory reaction. The mites in
both cases were put at the centre between two
treated areas (and some distance from each)
and their locations noted after a 5-minute period. This would indicate that the response of
an olfactory sense was being tested. However,
Phelan et al. (1991) stated that, Preliminary
observations of female mites walking in the
vicinity of honey bee extract revealed no orientation of the mites towards treated areas;
however, when placed in contact with a zone
treated with young-bee extract, the locomotory
tracts of the mite were dramatically dierent
from those of mites placed in hexane treated
zones. This would indicate that only a gustatory response was present and raises the questions as to why Phelan et al. (1991) used the
particular test methodology.

4.3. An alternative migration strategy

Phelan et al. (1991) examined the possible mechanism that determines the increased

susceptibility of young bees. It is widely accepted that mites only migrate to young bees
and that the susceptibility of young bees approaches zero when they are 4 to 5 days old.
This precise time period beyond which susceptibility is eectively zero is not determined by
increases in the grooming ability of the bees;
bees with little or no grooming ability have
also a susceptibility that approaches zero when
bees are 4 to 5 days old. Similarly, it is unlikely
to be determined by changes in the physiology of the bees. We know that most mites migrate at night when the bees are in a sedentary
mode, yet even highly susceptible bees have
few mites migrating after 4/5 days. It would be
expected that such precision in the age of the
bee would have to be determined by the mite,
rather than by a defensive mechanism of the
bee.
The results from these experiments also
raises the question of how the rapid reduction in bee infestation with age (Morgenthaler,
1930; Bailey, 1958; Lee, 1963; Giordani,
1977; Gary et al., 1989) arises in practice. It is
postulated that there are potentially two possible (rather than one) modes of mite migration.
The first is the generally accepted approach
whereby the mite would transfer to a neighbouring moving bee, test for suitability and
either accept or continue to quest for a suitable
bee (Sammataro and Needham, 1996). On the
assumption that contact between the bees in
the hive happens at random, it would be expected that a similar number of young bees
of a particular age in the range 0 to 20 days,
make contact with a host bee at the time that
a foundress female mite is questing. Hence
the mites may be seeking a particular chemical cue that identifies a very young bee and
must be leaving, for example, almost all of
the 520 day bees that they visit and must
also be moving towards the < 1-day old bees
even in the < 5-day old bees encountered. This
would appear to represent a high-risk strategy
for such a small animal that is in danger of
desiccation or being groomed o by the bees.
This indicates that the mite normally makes
several bee migrations before it can accomplish its task and the success rate is likely to be
low. External feeding by the mites on unsuitable hosts as observed in the experiments may

Host-seeking behaviour of Acarapis woodi

therefore be routinely practiced as an interim

measure to reduce the risk of desiccation but
the risk of being groomed o during transfer is
ever present. Given these costs, a less risky approach may have evolved whereby the migrating mite would evaluate the young bee before
transferring. This alternative mode is less risky
and given the rapid increase in mite infestation
that can take place in a colony (and hence high
transfer success rate) and the findings of Pettis
et al. (1992) that 85% of mite transfers take
place at night, this is likely to be the selected
mode of transfer. Pettis et al. (1992) considered that mites might detect movement of the
bees and wait until the host is motionless before initiating dispersal. This would also align
with a strategy of reducing risk during mite
migration.

ACKNOWLEDGEMENTS
The authors would like to thank the editor
and two anonymous reviewers for their helpful comments. Thanks to the members of Fingal
North Dublin Beekeepers Association and John &
Dorothy Stapleton for access to honey bee colonies.
Thanks also to Richard Jones, International Bee Research Association, for providing copies of old beekeeping papers.
Rle possible de signaux chimiques chez Apis
mellifera mellifera dans le comportement de recherche de lhte de lacarien Acarapis woodi.
Apis mellifera / Acarapis woodi / acarien / migration / nymphose / temprature de dveloppement
Zusammenfassung Chemische Reize beim
Wirtsfindeverhalten der Tracheenmilbe (Acarapis woodi) gegenber Honigbienen (Apis mellifera mellifera). Seit ber 100 Jahren wird die
Tracheenmilbe Acarapis woodi mit dem Zusammenbruch von Bienenvlkern in Verbindung gebracht. Es ist bekannt, dass die Milben von lteren
Bienen zu jngeren Wirtsstadien (meist weniger als
4 Tage alt) wechseln und dass die Anflligkeit eines Bienenvolkes gegenber einem Befall mit Tracheenmilben vor allem vom Putztrieb der Bienen
abhngt. In einem krzlich durchgefhrten Experiment konnte gezeigt werden, dass die Anflligkeit von Bienen, die sich bei einer niedrigeren Temperatur (30 C) verpuppten, signifikant erhht war.
Diese erhhte Anflligkeit konnte dabei nicht durch

577

einen geringeren Putztrieb erklrt werden. In der

vorliegenden Arbeit untersuchten wir, ob diese erhhte Anflligkeit mglicherweise durch den Einfluss chemischer Reize auf das Wanderungsverhalten der Milben verursacht wird (Abb. 3). Bienen,
die sich bei 30 C bzw. bei normaler Brutnesttemperatur von 34 C verpuppt hatten, unterschieden sich signifikant im Muster ihrer kutikulren
Kohlenwasserstoe. Verhaltensexperimente mit einem Wahl-Biotest zeigten allerdings, dass chemische Reize nicht die Prferenz der Milben fr Bienen, die sich bei 30 C verpuppt hatten, erklren
knnen (Tab. III). Wir schlieen daraus, dass andere Faktoren wie zum Beispiel eine durch niedrige Verpuppungstemperaturen verursachte verringerte Mobilitt oder Wahrnehmungsfhigkeit der
Bienen fr die erhhte Anflligkeit verantwortlich
sind. Unsere Untersuchung zeigt auch, dass die Milben eher durch Geschmack als durch Geruchsreize in ihrem Verhalten beeinflusst werden. Es wird
ein neues Modell fr die Milbenwanderung zu den
jungen Bienen (<4 Tage) vorgestellt, bei dem der
Wirtswechsel und damit die Verbreitung des Parasiten mit einem geringeren Risiko fr die Muttermilben verbunden sind.
Apis mellifera / Acarapis woodi / Tracheenmilbe /
Milbenwanderung / Verpuppungstemperatur

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